Bacteria of the genus, Clostridium, secrete seven highly potent botulinum neurotoxins (BoNTs) (serotypes AG), which pose a significant threat as weapons of bioterror. Each toxin presents a highly lethal protease that enters human motor neurons and proteolytically inactivates a protein required for neurotransmission. The protein substrates destroyed by BoNT proteases have been identified; however, the amino-acid motifs that render these proteins susceptible remain largely uncharacterized, primarily because a rapid cell-based assay is not available to dissect the unusually long amino acid stretches recognized by the BoNT proteases. We have overcome this problem by developing a novel cell assay for BoNT serotype B, which brings the powerful tools of yeast genetics to the study of a BoNT protease. With this assay, yeast-colony formation on an agar plate is available to monitor the lethal action of BoNTB. In Aim 1, we will employ the assay to genetically select large numbers of mutations that protect cells from BoNTB substrate cleavage and, consequently, toxin-induced lethality. Substrate amino acids targeted by these mutations will, thus, molecularly define the BoNTB substrate motif. In Aims 2 and 3, we will expand our experimental approach by developing assays that can monitor the proteolytic activities of the remaining six BoNT serotypes in yeast. The assays will then be utilized to dissect the substrate specificity of each toxin. By utilizing novel yeast assays for BoNT intracellular proteases, our study will provide the most complete pictures to date of the substrate motifs recognized by BoNTs and lead to the development of important experimental cell systems, which could be used to select and identify the next generation of anti-BoNT therapeutics.